Exosomes are microvesicles of a size ranging between 30-120 nm, actively secreted through an exocytosis pathway normally used for receptor discharge and intercellular cross-talk. Exosomes may be detected in cell culture supernatants and some body fluids, following multistep ultracentrifugation. In addition to major hisotcompatibility complex proteins (MHCI, MHC II) and proteins involved in antigen presentation, exosomes may carry membrane and cytocolic proteins involved in many cellular functions. Exosomes are secreted under specific physiological conditions from different cell types such as dendritic cells (DC), lymphocytes, mast cells and epithelical cells. This process leads to the formation of basket-like cellular reservoirs that contain these multifusion-derived microvesicles, also called Multivesicular Bodies (MVB). This process has been resolved through utrastructure observations, particularly in normal cells. However, through immunoelectron microscopy and western blot analysis of exosome preparations it has been shown that these microvesicles co-express markers of different intracellular vacuoles, such as early endosomes (e.g. Rab5), lysosomes (e.g. CD63, CD81, LAMP-1) and late phagosomes (Rab7), but also some other more cell-specific proteins (e.g. MHC class III antigens).
Release of exosomes from tumor cells is dramatically higher than from normal cells, and it is often associated with immunosuppressive effects. Tumor derived exosomes are in many aspects comparable to the exosomes of normal cells, except for the expression of some tumor markers, such as CEA for colonic carcinoma, MART-1 or gp-100 for melanoma. Moreover, the origin of tumor exosomes seems to be in many aspects dissimilar to that of normal exosomes. This is probably because malignant cells have more dynamic vesicle traffic from the cytoplasm to the extracellular space and vice versa.
The role of tumor exosomes in cancer progression is a recently emerged reserach area. Inital data suggests that these organelles act as carriers of tumor antigenic material for DC-mediated T cell crosspriming. Such results give support to clinical attempts to use tumor exosomes as anti-cancer vaccines. Growing evidence concerning a vast array of suppressive effects exerted by these microvesicles on different components of the immune system is clearly supporting the involvement of tumor exosomes in disease progression. In particular, it has been recently shown that exosomes secreted by human tumor cells of various origins are able to induce apotposis in activated T cells, through the expression of death ligands (e.g. FasL, TRAIL), inhibit NK functions and promote the generation of myeloid-derived suppressor cells from normal monocytes. These data, together with the reproducible evidence that exosomes of likely tumor origin can be abundantly found in plasma and nonplastic effusions of cancer patients, support a role of tumor exosomes in molding host microenvironment to allow tumor cells growth and progressions.
Given the increasing understanding of the role of exosomes in cancer progression and the fact that there is an increasing need to find new cures, improve diagnostics and follow up of malignancy and growth of tumors, there is accordingly a need for methods and tools to detect and measure exosomes in human fluids. The fact however is, that methods that are currently used to detect exosomes are either not quantitative (TEM) or only poorly quantitative (WB). Although flow cytometry has been used to quantify exosomes, this method does not allow a precise measurement that the researchers need. In fact, while FACS (Fluorescence Activated Cell Sorter) analysis is a suitable method to quantify cells, even of small size, but it is not suitable to quantify the amount of such small vesicles (i.e. 50-100 nm). Moreover, the rough measurement of total mean fluorescence does not allow a precise quantification on how many microvesicles are present in the given sample. Furthermore, FACS analysis does not allow simultaneous analysis of different samples. Therefore, there is a need for a method to detect and quantify exosomes from small amounts of body fluids. Moreover, given the fact that early diagnosis of cancer is essential for disease treatment, there is a need for potential tumor markers and prognostic factors.